CHM 341 Suroviec Fall 2016 I Nucleotides Nucleic Acids and Bases Bases Planar aromatic heterocyclic Purine 2 rings Pyrimidine 1 ring Adenine A Guanine G Thyamine T ID: 709752
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Slide1
Nucleic Acids, DNA, RNA and Protein Synthesis
CHM
341
Suroviec
Fall
2016Slide2
I. Nucleotides, Nucleic Acids and Bases
Bases
Planar, aromatic, heterocyclic
Purine (2 rings)Pyrimidine (1 ring)
Adenine (A)
Guanine (G)
Thyamine (T)
Cytosine ( C)
Uracil (U)Slide3
B. Nucleosides
Ribonucleotides
sugar = ribose
DeoxyriboneculeotideSugar = 2´-deoxyriboseSlide4
C. Nucleotides (total molecule)
Have a phosphate on carbon #5
Can have up to 3 phosphates
Monophosphate (NMP)Diphosphate (NDP)
Triphosphate (NTP)Where N is any one of the nucleic acidsSlide5
II. Nucleic Acid Structure
Can be found singly
Most often found in a polymer
DNA (or RNA) polymerizes 5´ phosphate to 3´ OH
Makes phosphodiester bondPolymer of non-identical residues has a property that individual monomers do not.Slide6
A. Base composition of DNA
1940’s Erwin Chargaff discovered that when measuring the amount of each base A = T and G = C. Lead to Chargaff’s rules
.
Maurie
Wilkins and Rosalind Franklin made and X-ray that indicated that DNA was helical in nature
Watson and Crick took this data and other material that hinted that DNA stacked to propose that DNA was double stranded.Put the bases together in such a way so that the complimentary H-bonds were formed and the width of the base pairs would be similar.Slide7
Characteristics of DNA model
DNA strands run in opposite directions (antiparallel)
Sugar phosphate backbone is found on outside, bases inside and pair up
Each base is H-bonded with a base on the opposite strand with the same number of H-bonds
A complete turn takes 34 Å
and has 10 bases per turn2 helical polynucleotide chains coiled around a central axis (diameter = 20 Å)DNA strand is quite stiff and will not bend much around the axisSlide8
- DNA
Helix is right handedSlide9
III. Overview of Nucleic Acid Function
Carries genetic info
Directs protein synthesis
Double stranded nature allows for easy replication
DNA replicationW-C model allows each DNA strand to act as template for replication
2 hypothesis for replication came forth:Conservative: where the parental DNA strand retains both old stands and creates new ds DNASemi conservative: where the created DNA has one old strand and one new strand. Shown to be how DNA replicatedSlide10
DNA, RNA & Protein Synthesis
DNA directs its own replication and is also transcribed into RNA.
RNA then translates into proteins.
CENTRAL DOGMA of MOLECULAR BIOLOGY
Transcription: transferring into from DNA --> RNATranslation: transferring info from RNA --> proteinsSlide11
IV. Replication
Involves 20+ proteins
Helicases: opens the double strand, splits the strands apart starting at replication fork rich in A-T
SSB: bind to the single strand DNA
stablizing itPrimase: adds short stretches of RNA and allows the the DNA polymerase to start.
DNA polymerase I: catalyzes the addition of deoxynucleotides to the chain
DNA Polymerase I & III:
add DNA
with high fidelity to
the
newly growing DNA strand.
Ligase: closes up gaps
in
the DNASlide12
DNA polymerase I
DNA polymerase I catalyzes addition of a addition of dNTP to chain
Requires
dATP, dGTP,
TTp, dCTP and Mg2+Elongation occurs 5´ to 3´ where 3´ hydroxyl bind to the new
deoxyribonucleotideDNA polymerase is a “template directed enzyme”Slide13
DNA polymerase III
Adds nucleotides to the 3´ end of the chain
New strand reads 5´ to 3´
Needs a primer with free 3´ hydroxyl group to start addition of new DNA
The strand is going to be started with a RNA primer that is later removed and replaced
The incoming dNTP first forms an appropriate base pair and then the DNA polymerase III links the incoming bases togetherBinds complementary DNA nucleotides starting at the 3´ end of the RNA primer at a rate of 1000/secondMakes a mistake 1/108Slide14
IV. Replication
Opening of the DNA
Double stranded DNA is opened by helicase
Kept open by SSB
Exposed DNA bind DNA polymerase III and RNA synthesizing protein primaseThis makes the replication forkSlide15
IV. Replication
Leading strand synthesis begins with synthesis of primase of short RNA primer
dNTPs are added by DNA polymerase III
Continously
added to this strand toward the forkSlide16
Replication
Lagging strand synthesis is done in short bursts
Needs multiple RNA primers
Synthesized in opposite direction of the fork
DNA primase moves 5’ to 3’ and makes RNA primer to which DNA is then added by DNA polymerase IIISlide17
Replication
Keeping the DNA sequence correct is important: 1
mispair
per 109 base pairs
Polymerase reaction occurs in 2 stagesIncoming dNTP base pairs with the template while enzyme is open catalytically inactivePolymerization only occurs after polymerase has closed around base pair which positions residuesSlide18
Transcription
DNA is in the nucleus
Protein synthesis takes place in the ribosome
RNA is the intermediate
Cells contain 3 types of RNARibosomal RNA (rRNA)Transfer RNA (tRNA)
Messenger RNA (mRNA)Slide19
RNA polymerase
RNAP couples together the ribonucleotide triphosphates on DNA templates
Builds RNA in the 5’
3’ direction (reads the DNA in the 3’ --> 5’ direction)Slide20
RNA polymerase
3’ hydroxyl group attacks the triphosphate
Creates phosphodiester bond
Releases PPi
Does not need a primerSlide21
RNA polymerase
Initiation of RNA synthesis occurs only at promoters
Usually starts at GTP or ATP
New RNA strand base pairs temporarily with DNA template to form DNA/RNA template
DNA must unwind then rewind Template strandNontemplate
strand or coding strandSlide22
RNA polymerase
RNA polymerase lacks ability to proof read
No 3’--> 5’ exonuclease activity
One error in 104 ribonucleotides addedSlide23
Post transcription of RNA
In Eukaryotes RNA is further modified
mRNA undergoes gene splicing where introns are removed and exons are rejoined
5’ obtains a cap3’ gets polyA
tailSlide24
Characteristics of RNA
Contains AUGC
Uracil is less “energy expensive”
Normally single stranded
Has –OH on 2’ carbon of riboseSeven roles of RNA
mRNA – carries DNA code to make proteinsrRNA – forms complex of 2/3 RNA, 1/3 protein to form protein in ribosometRNA – carries the amino acids to the mRNAsnRNA – helps splice exonsRibozymes – RNA capable of catalytic activityAntisense RNA – act to bind RNA to stop translationViral RNA – carry hereditary informationSlide25
Translation
mRNA to proteins
Need mRNA, ribosome and
tRNAmRNA is produced from DNAmRNA read from ribosomes and
tRNASlide26
Ribosome
Large protein/RNA complex
2 units (large/small)
Synthesis begins at start codon near 5’ end
Smaller unit (usually has tRNA bound) binds to AUG codon on mRNA binds to large subunit
Large unit then bindsLarge unit has 3 tRNA binding sites (APE)A: aminoacyl-tRNAP:peptidyl-tRNAE: free-tRNASlide27
Initiation
AUG signals the beginning of polypeptide chains
Read the code off of the mRNA and translate into amino acids
One start codon3 stop codonSlide28
tRNA
Read the code on the mRNA and translate into the correct amino acid
Acceptor stem
5’ terminal nucleotide and 3’ terminal nucleotide (-OH group where amino acid binds)
3’end always has CCA sequenceSpecific linkage is catalyzed by amino acyl-tRNA
synthetase (tranferase). Anticodon recognizes the complementary codon on the mRNASlide29
Aminoacylation
Process of adding an aminoacyl group to a compound
Produces
tRNA molecules with their CCA 3’ ends covalently linked to an amino acidAminoacyl
tRNA synthetase (one specific for each amino acid)Needs ATP to drive the reactionSlide30
Initiation and Elongation
mRNA bearing the code for the polypeptide binds to the small ribosome unit
Aminoacyl-
tRNA then binds followed by larger ribosomal unitAminoacyl-
tRNA base-pairs with mRNA codon AUG to start the polypeptide
Chain is elongated by addition of amino acidsAdded by individual tRNAPolypeptides are grown from amino-terminal end to carboxyl-endSlide31
Elongation
mRNA passes through ribosome
AUG is held in P site
2nd amino acid binds in the A site
Make peptide bondRibosome then moves toward 3’ end using GTP and leaving A site open